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In plant metabolism glycerol phosphate must be produced for the synthesis of various phospholipids, sulfolipids, galactolipids, and triglycerides. The metabolic reaction in leaf tissue of higher plants for glycerol phosphate synthesis is catalyzed by a DHAP2 reductase which has been partially purified from spinach leaves and castor bean endosperm, and which catalyzes the reduction of DHAP at pH 7.0 using NADH as the reductant (6,17 No. 12290. 2Abbreviations: DHAP, dihydroxyacetone phosphate; ME, mercaptoethanol; PVPP, polyvinylpolypyrrolidone. metabolism must be highly controlled, as is expected of compounds and enzymes at metabolic branch points.A number of free living algae produce glycerol as a major product of photosynthesis rather than accumulate starch or sucrose. Zooxanthellae, symbiotic, unicellular algae in the polyps of reef building corals and other marine invertebrates, excrete to their host up to 40% of their photosynthate as glycerol (14, 15).The halotolerant alga, Dunaliella tertiolecta (5,20) and Chiamydomonas (1 1)

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The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de noro pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.Numerous in vivo studies have established that the pyridine moiety of quinolinic acid, nicotinamide, and nicotinic acid is directly incorporated into the alkaloids ricinine and nicotine produced by the castor bean plant and the tobacco plant, respectively (7,11,20,25,26). In plants, one metabolic pathway leading to the formation of pyridine nucleotides is the de novo pathway, involving initially the condensation of aspartic acid and glyceraldehyde-3-P or closely related metabolites. The condensation product of aspartic acid and glyceraldehyde-3-P undergoes a series of reactions leading to the formation of quinolinic acid which is then converted to pyridine nucleotides. The only known enzyme in this pathway is quinolinic acid phosphoribosyltransferase, which catalyzes the conversion of quinolinic acid and phosphoribosylpyrophosphate to nicotinic acid mononucleotide (7,15). The latter is then converted to NAD via nicotinic acid adenine dinucleotide (16). By means of the pyridine nucleotide cycle, the NAD so made in plants is eventually broken down into nicotinamide and nicotinic acid, which in a cyclic process can also be converted to nicotinic acid mononucleotide and subsequently to NAD (6,22). The known reactions of the de novo synthesis of pyridine nucleotides and the reactions of the pyridine nucleotide cycle are shown in Figure 1. Because quinolinic acid is easily converted to nicotinamide and nicotinic acid (7), and because all three compounds are excellent precursors of the pyridine alkaloids, ricinine and nicotine, the pyridine nucleotide cycle could lie between the de novo pathway for pyridine nucleotide biosynthesis and the pyridine alkaloids, as first s...

The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH4)2SO4. Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C3 photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis. tors are so effective, no activity had earlier been detectable in crude leaf homogenates or chloroplast preparations (3, 6). Enzyme activity could only be measured by first precipitating a protein fraction with 35 to 70% saturated (NH4)2SO4 followed by dialysis. However, it has now been possible to detect the DHAP reductase in a crude homogenate after centrifugation at 110,000g for 1 hr and addition of BSA to remove most lipids and membranes and addition of DTI to stimulate the chloroplastic form.In addition to NADH:DHAP reductase, thioredoxin regulates the activity of a number of chloroplast enzymes, and, therefore, stimulation of the DHAP reductase isozyme in the chloroplast by thioredoxin is consistent with previous findings with other chloroplast enzymes. An increase in the activity of DHAP reductase would lead to increased production of glycerol phosphate, which in turn would be used for lipid synthesis or might yield glycerol by dephosphorylation.The present study indicates that Fru 2,6-P2 stimulates the cytoplasmic DHAP reductase, which could also result in increased lipid synthesis or amounts of glycerol in the cytoplasm. Recent investigations (7-11) have indicated that Fru 2,6-P2 also inhibits cytosolic Fru 1,6-P2 phosphatase to block the formation of sucrose and promote glycolysis. Thus, regulation of the cytosolic DHAP reductase isozyme appears to be another cytoplasmic systems that is regulated by Fru 2,6-P2. MATERIALS AND METHODSThere are two forms of sn-3-phosphoglycerol dehydrogenase (EC 1.1.1.8) or NADH:DHAP2 reductase in leaves (3,6). In the leaf, the isozyme in the chloroplast...

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